Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus

Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-β-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb’ebony and Gb’tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb’ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb’tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb’ebony and Gb’tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb’ebony and Gb’tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.

Introduction Body color is one of the most diversified features in insect morphology and is involved in camouflage, aposematism, and other processes [1,2]. In addition to the evolutional diversity of body color patterns observed between and within species such as flies [2], it changes throughout the developmental stages of insects. Nymphs and adults of Hymenopus coronatus (known as the Orchid Mantis) show distinct color patterns. The first instar nymphs show a black-red color pattern, the second to last instar nymphs show a flower-like pattern, and the adults show a brown-white color pattern [3], and these color patterns are adaptive to the surrounding environment.
The cricket Gryllus bimaculatus (common name, two-spotted cricket) is a model hemimetabolous insect species [4]. Crickets, especially G. bimaculatus, have recently gained attention as a biological material for protein production [5,6]. Studying gene functions in this insect is advantageous because of the availability of genomic data [7] and genome-editing technologies, including clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins 9 (Cas9) [8,9]. This cricket species exhibits stage-specific body color patterns of a black-yellow-brown combination during postembryonic development; thus, it is suitable for analyzing the molecular mechanisms of the body color transition between developmental stages.
During the pigmentation process, the exoskeleton cuticle acquires coloration through a combination of melanin and sclerotin pigments. Previous studies in insects such as Drosophila melanogaster [2] and Tribolium casteneum [10] have demonstrated that dopamine is the core substrate in the model pathway of melanin and sclerotin biosynthesis. As shown in Fig 1, the biosynthesis of melanin and sclerotin starts from tyrosine, which is converted into L-3,4-dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase (pale), the rate-limiting enzyme in this pathway. DOPA is converted into dopamine by DOPA decarboxylase (ddc), and dopamine is then used as a substrate to produce dopamine-melanin, a black pigment produced by the dopamine chrome conversion activity of the Yellow protein (yellow) and the phenol oxidase activity of Laccase2 (lac2). Excess dopamine is metabolized into N-β-alanyl dopamine (NBAD) and N-acetyl dopamine (NADA) by NBAD synthase (ebony) and dopamine N-acetyltransferase (nat), respectively. NBAD and NADA are also metabolized by Laccase2 to synthesize yellow NBAD sclerotin and colorless NADA sclerotin, respectively. In addition to these enzymes, this pathway may include NBAD hydroxylase (tan), which degrades NBAD into β-alanine and dopamine. Sclerotin also functions as a sclerotizing agent, i.e., it provides rigidness to insect cuticles [11,12]. Previously generated G. bimaculatus lac2 mutants with genome-editing displayed transparent cuticles and died within a few days after hatching [13], indicating that, similar to other insects, cuticle pigments in crickets are mostly melanin and sclerotin.
The importance of ebony and tan for cuticle pigmentation has been reported in other insects. On the one hand, knockout and/or knockdown of ebony leads to a black body color in the holometabolous insects D. melanogaster, T. casteneum, and Henosepilachna vigintioctopunctata, and in the hemimetabolous insects Periplaneta americana and Oncopeltus fasciatus [14][15][16][17]. In these insects, ebony systemically determines body color. On the other hand, tan is a suppression factor during NBAD synthesis. Mutations in tan reduced melanization in D. melanogaster [18]. In O. fasciatus, tan knockdown reduces the level of melanin pigmentation in a limited area [17]. The function of tan on the body color of insects likely varies among species.
In this study, using CRISPR/Cas9, we generated homozygous knockout mutants of G. bimaculatus (Gb) ebony and tan, which we used to analyze the in vivo functions of these genes. Additionally, we analyzed the changes in the expression levels of the Gb'ebony and Gb'tan transcripts in wild-type crickets and compared them with body color patterns. Our results provide evidence for the importance of Gb'ebony and Gb'tan in generating body color variation in the nymph and adult stages of the cricket.

Identification of Gryllus ebony and tan
Using the sequences of ebony and tan from other insects as references, we searched for similar sequences in the G. bimaculatus genome (GenBank: GCA_017312745.1). The resulting information was used to clone 2,595 bp of Gb'ebony cDNA, including the full-length coding sequence. Gb'ebony was encoded on scaffold 146 of the G. bimaculatus genome throughout 15 exons (Fig 2A). The deduced Gb'Ebony protein consists of 864 amino acids with a molecular weight of 89,890. The primary structure of Gb'Ebony contains an AMP-binding domain located at 34-448 amino acids in the protein sequence. The results of phylogenic analysis of the amino acid sequences of Ebony proteins from Gryllus and other insects (aminoadipatesemialdehyde dehydrogenase (Aasdh) from D. melanogaster, a paralog of Drosophila ebony, was included as an outgroup) show that Gb'ebony is an ortholog of the ebony gene of other insects (Fig 2B).

Fig 1. Pathway of melanin and sclerotin biosynthesis in insects.
Dopamine, which is synthesized from tyrosine by the activity of TH and Ddc, becomes three pigments. Dopamine-melanin is produced by the activity of Yellow and Lac2. Excess dopamine is metabolized into NBAD and NADA by the activity of Ebony and NAT, respectively, to produce two types of sclerotins: yellow NBAD sclerotin and colorless NADA sclerotin. This pathway also includes Tan, an NBAD hydroxylase, which may coordinate the melanization level with Ebony. The metabolic pathway depicted was modified from Arakane et al. (2009) [10]. Intermediate products and pathways of DOPA-melanin biosynthesis have been omitted to demonstrate Dopamine-related pigmentation. Enzymes and metabolites are colored red and black, respectively. TH, Tyrosine hydroxylase; Ddc, DOPA decarboxylase; Ebony, NBAD synthase; Tan, NBAD hydroxylase; NAT, dopamine N-acetyltransferase; Yellow, Yellow protein. https://doi.org/10.1371/journal.pone.0285934.g001 We also cloned 1,455 bp of Gb'tan cDNA, which includes the full-length coding sequence. Gb'tan was encoded on scaffold 11 of the G. bimaculatus genome throughout 7 exons (Fig 2A). The deduced Gb'Tan protein consists of 393 amino acids with a molecular weight of 43,118. The primary feature of Gb'Tan is a 6-aminopenicillanic acid acyltransferase domain (Pfam03417) located at 127-376 amino acids in the deduced protein sequence. The results of  the phylogenic analysis of the amino acid sequences of Tan from Gryllus and other insects (isopenicillin N-acyltransferase (IAT) from fungi Penicillium chrysogenum, the ancestral protein of insect Tan, was included as an outgroup) show that Gb'tan is an ortholog of the tan gene of other insects (Fig 2B). The sequences of Gb'ebony and Gb'tan were deposited into the DNA Data Bank of Japan under accession no. LC733201(Gb'ebony) and LC733202 (Gb'tan).

Expression profiles of Gb'ebony and Gb'tan transcripts from the embryo through adult stages
To examine the relationship between the Gb'ebony and Gb'tan transcripts and cuticle pigmentation in the cricket, we analyzed the expression profiles of these genes from the embryo through the adult stages. Gb'ebony transcripts were detected 7 days after egg laying, and transcript levels increased immediately after the first instar nymphs hatched (Fig 3A). The expression level of the Gb'ebony transcript decreased rapidly after the completion of pigmentation a few hours after hatching and molting (compare the filled and unfilled points at D1 in the first to third instars enclosed with a red dotted frame in Fig 3A). The expression level before and after pigmentation in first instar nymphs showed a significant difference (P < 0.01). The expression level decreased after the peak expression in first instar nymphs and increased again in fifth instar nymphs. In the fifth instar and subsequent stages, a high level of expression immediately after molting and a subsequent decrease in expression were observed, and the peak expression tended to increase stage-to-stage. In the later stages, from the sixth to eighth instars, the expression levels of Gb'ebony transcripts varied greatly between individual crickets. At the seventh instar stage, expression in males tended to be higher than that in females. Gb'ebony expression was undetermined (Ct > 35) in several samples obtained from day 3 of the fifth, seventh, and eighth instars. Additionally, the expression level of Gb'ebony in adult males was significantly higher than that in adult females.
Gb'tan transcripts were detected 5 days after egg laying, and their levels increased immediately after hatching and each molting ( Fig 3B). Gene expression decreased rapidly with the completion of melanin pigmentation in a few hours on day 1 of the first to third instar nymphs (compare the filled and unfilled points at D1 in the first to third instars enclosed with a red dotted frame in Fig 3B). There was a significant difference in gene expression, especially before and after the completion of melanin pigmentation on day 1 of the first (P < 0.05) and second (P < 0.01) instar nymphs. The highest expression level of Gb'tan occurred in first instar nymphs and gradually decreased to a peak of expression with each developmental stage. We observed no sex differences in the expression levels of Gb'tan transcripts.

CRISPR/Cas9-based generation of Gb'ebony and Gb'tan homozygous knockout mutants
The in vivo functions of Gb'ebony and Gb'tan were investigated by generating homozygous knockout mutants using CRISPR/Cas9. We designed two guide RNAs (gRNAs) that bind to the third exon of Gb'ebony and one gRNA that binds to the second exon of Gb'tan (Fig 2A). Although only a single gRNA was used for Gb'tan knockout, its specificity was verified with the BLASTN program to search for matches within the Gryllus genome (GenBank: GCA_017312745.1) (S1 Fig). After injecting the Cas9-gRNA complex into the eggs, sitedirected mutagenesis was detected using a mismatch-specific endonuclease, Guide-it Resolvase (Takara Bio, Shiga, Japan). As shown in Fig 4A, the DNA bands were digested when the PCR products from G0 eggs were incubated with the endonuclease. PCR products from the wild-type were not cleaved by the endonuclease. Raw data from this experiment can be found in S1 Raw images. Furthermore, sequencing analysis confirmed the introduction of mutations in the gRNA target region of G0 eggs (S2 Fig). and Gb'tan (B) transcripts in whole embryos (D; day after egg laying) and the whole body of nymphs and adults (D; day after hatching or molting) were analyzed by RT-qPCR. Gb'β-actin was used as an internal control gene. Black, blue, and red lines indicate unsexed, male, and female crickets, respectively. Filled and unfilled points indicate pigmented and unpigmented crickets, respectively. The scale on the X-axis indicates one day. The first to third instars showed complete pigmentation within a few hours after hatching and molting. Gene expression in unpigmented and pigmented crickets on day 1 of each stage was analyzed using cDNA samples derived from crickets within 1 and 2-15 h, respectively, after hatching or molting (enclosed with a red dotted frame). Relative expression levels were calculated based on the amounts of transcripts in the first instar nymphs immediately after hatching. The data presented are the mean and SD (N � 3). The lower sides of the error bars were omitted. The asterisks (*) and (**) mean P < 0.05 and P < 0.01, respectively, based on Student's t-test. Asterisks for the first to third instars are shown for significant differences in gene expression levels between unpigmented and pigmented crickets on day 1 of each stage. Asterisks from the seventh instar onward are indicated when there was a significant difference in gene expression between males and females. https://doi.org/10.1371/journal.pone.0285934.g003 We reared G0 insects and crossed them with wild-type crickets. The resulting eggs were genotyped, and the G1 heterozygous offspring of Gb'ebony and Gb'tan were sequenced. Male Gb'tan transcription. RNA was extracted from the whole body of day 1 seventh instar nymphs of the wild-type and Gb'tan mutants within 1 h of molting and subjected to RT-qPCR. Data are presented as the mean value ± SD obtained from four biological replicates and three technical replicates. The asterisks (**) mean statistical significance at P < 0.01 in a Student's t-test.
https://doi.org/10.1371/journal.pone.0285934.g004 and female mutants with the same type of frameshift mutation were isolated and crossed. Further sequence analysis of the gRNA-targeted region in the G2 offspring showed that the Gb'ebony frameshift mutants harbored a homozygous 5 bp deletion (Gb'ebony cr1 ) and a 4 bp insertion (Gb'ebony cr2 ) ( Fig 4B). Meanwhile, the Gb'tan frameshift mutant carried a homozygous +1 bp insertion/deletion (Gb'tan cr1 ) (Fig 4B). Only one Gb'tan mutant strain was generated in this study, but mutations in off-target sites in Gb'tan crRNA1 (S1 Fig) were not observed in sequence analysis (S3 Fig). To confirm whether the functions of Gb'ebony and Gb'tan in the mutants were completely disrupted at the protein level, we performed immunoblotting with polyclonal antibodies against the Gb'Ebony and Gb'Tan proteins. The anti-Gb'Ebony antibody recognized a 90-kDa protein in the wild-type, which corresponds to the molecular mass of the Gb'Ebony protein (95,963, calculated based on the estimated full-length coding sequence) (Fig 4C). However, the Gb'Ebony protein was not detected in the Gb'ebony cr1 and Gb'ebony cr2 mutants. Immunoblotting with anti-Gb'Tan detected a 25-kDa protein in the wild-type (Fig 4C). This protein band was not detected in the Gb'tan cr1 mutant. However, the size of this band was smaller than the expected molecular mass of the Gb'Tan protein (43,118). The primary structure of the Gb'Tan protein shows homology with the acyl-coenzyme A:6-aminopenicillanic acid acyltransferase, a member of the peptidase C45 family. IAT from P. chrysogenum, which also belongs to the peptidase C45 family, is produced as an inactive precursor and matures through posttranslational self-cleavage at the Gry102-Cys103 peptide bond, resulting in 11 and 29 kDa α-and β-subunits, respectively [19]. In D. melanogaster, the 45-kDa Tan pre-protein is self-processed at Gry121-Cys122 into 30-and 15-kDa α-and β-subunits, respectively [20]. We confirmed that the self-cleavage site in D. melanogaster Tan was conserved in the Gb'Tan protein at Gry121-Cys122 (S4 Fig). Cleavage of the Gb'Tan protein at the same site should produce a 29.7-kDa protein with an active 6-aminopenicillanic acid acyltransferase domain (Pfam03417). This result suggests that the 25-kDa Gb'Tan protein is probably the mature form. To further validate the Gb'tan knockout, the transcript levels of Gb'tan were analyzed by RT-qPCR. The results showed that the amount of Gb'tan transcripts in the Gb'tan mutant line was significantly reduced to about one-tenth of that in the wild-type (P < 0.01, Fig 4D), demonstrating that mutagenesis was effective at the transcriptional level. Overall, these results indicate that the Gb'ebony and Gb'tan homozygous knockout mutants completely lost their functions.

Phenotypes of homozygous Gb'ebony knockout mutants
We predicted that the Gb'ebony knockout mutation would cause a loss of yellow pigment because the gene encodes NBAD synthase, which is required for the synthesis of yellow sclerotin. Adult Gb'ebony cr1 mutants had yellow-colored regions that were fully darkened compared to the wild-type, especially in the legs and forewings (Fig 5A). In the wild-type, the central region of the forewing was lightly transparent. This region in the Gb'ebony mutants was colored black and opaque. Fig 5B shows a region surrounded by two wing veins (arrowhead) and the upper edge of the hindwing that were colored black (Fig 5B). These black pigments may be dopamine-melanin.
G. bimaculatus is commonly known as the two-spotted cricket, a name derived from the spotted pattern on its forewing. In wild-type adults, the color of the anterior side of the forewing was lighter than that of the other side, producing a spotted pattern (Fig 5A). In Gb'ebony mutants, surprisingly, this pattern was colored white (probably due to colorless NADA sclerotin), although other areas of the forewing were dark. These results indicate that Gb'ebony is essential for the synthesis of yellow pigments, and its knockout causes systemic darkening of body color. In the nymphal stages, the dorsal side of the thorax segments 1 and 2 and the tail surface in the first instar nymphs were yellow (Fig 5C and 5C'). In Gb'ebony mutants, these regions were melanized. In the second to fourth instar nymphs, the change in the body color of the Gb'ebony mutants was limited because the wild-type originally has a dark black body color. Starting from the sixth instar stage, the body color of the wild-type became increasingly yellow until the eighth (ultimate) stage. In contrast, the yellow pigment of the Gb'ebony mutants was lost during these stages. These results indicate that Gb'ebony is required for the systemic determination of body color. We also analyzed the Gb'ebony cr2 strain, which was generated using a different gRNA (crRNA2) than that used in the generation of the Gb'ebony cr1 strain. The Gb'ebony cr2 mutants exhibited systemic darkening of body color at all stages (S5

Phenotypes of homozygous Gb'tan knockout mutants
Gb'tan encodes NBAD hydroxylase, which degrades NBAD into dopamine and β-alanine, a function opposite to that of NBAD synthase, which is encoded by Gb'ebony. Therefore, we predicted that the Gb'tan knockout would enhance NBAD yellow sclerotin synthesis and reduce melanin pigmentation. However, as seen in Fig 6A, the change in the body color of adult Gb'tan cr1 mutants was comparable to that of the wild-type. Still, in the isolated male forewing of the adult Gb'tan cr1 mutants, the melanin pigmentation level was slightly reduced, and light transparency was enhanced (Fig 6B), indicating that the synthesis of dopamine-melanin was inhibited by the Gb'tan knockout. The body color of Gb'tan mutants at the seventh instar appeared remarkably lighter than that of the wild-type ( Fig  6C). To quantify the color difference between the wild-type and Gb'tan mutants, we measured the mean grayscale intensity, consisting of 256 tones of color gradients, with 0 and 255 indicating white and black, respectively, at the penultimate nymph stage. A lower and higher intensity value indicates a darker and lighter body color, respectively. In males, the mean intensities of the wild-type and Gb'tan mutants were 77 and 96, respectively, significantly different (P < 0.01) (Fig 6D). In females, the mean intensities of the wild-type and Gb'tan mutants were 84 and 96, respectively ( Fig 6D). This also showed a significant difference (P < 0.05), but less different than in males. These results indicate that Gb'tan is essential for proper melanin pigmentation in limited developmental stages and tissues, and its knockout enhances the synthesis of NBAD yellow sclerotin.

Discussion
Insect dopamine is an important substrate for the biosynthesis of pigments such as dopaminemelanin, NBAD sclerotin, and NADA sclerotin. In this study, we focused on the function of G. bimaculatus ebony and tan, which are involved in dopamine metabolism.

The expression of the Gb'ebony and Gb'tan transcripts during hatching and molting
We first examined the expression profiles of Gb'ebony and Gb'tan during development to assess their relationship with cuticle pigmentation. After hatching and molting, crickets (re) construct body color on the cuticle within a few hours. During these periods, the expression levels of Gb'ebony and Gb'tan were elevated and then decreased after pigmentation was completed (Fig 3). These results indicate that Gb'ebony and Gb'tan are involved in cuticle pigmentation in crickets. Similarly, in H. vigintioctopunctata (Hv), the expression of Hvtan and Hvebony is upregulated before and/or immediately after molting [16].
Insect metamorphosis is rigidly regulated by two molting hormones: juvenile hormone (JH) and 20-hydroxyecdysone (20E) [21]. High levels of both 20E and JH occur when nymphs are molting, whereas only the 20E titer is high during metamorphosis into the adult stage. In G. bimaculatus, the levels of JH and Gb'E93 (an ecdysone-induced protein) increase periodically during molting [22,23], following similar patterns as that of Gb'ebony and Gb'tan. Additionally, in Bombyx mori, BmDdc is expressed in response to exogenously administered phytogenous ecdysteroids [24]. Overall, these results indicate that pigment metabolism genes, including Gb'ebony and Gb'tan, are regulated by the molting hormone signal cascade.
Molting is accomplished in three phases: the preparatory, cuticle induction, and ecdysial phases [21]. These phases are regulated by 20E; for example, the initiation of the cuticle induction phase requires a high 20E titer. During the cuticle induction phase, Drosophila express the ecdysone-induced transcription factors E74A and E75B. In Tribolium, E75 is required for adult metamorphosis, and its RNAi knockdown causes an enhanced expression of Th, the gene encoding tyrosine hydroxylase, which is the initial enzyme in dopamine synthesis [25]. The expression of E75 is suppressed by JH-mimic treatment. These findings from other studies suggest that cuticle pigmentation is controlled by 20E and JH upon molting. However, the mechanism that regulates Gb'ebony and Gb'tan during molting remains unknown.

Role of Gb'ebony in cuticle pigmentation
Gb'ebony encodes NBAD synthase, which catalyzes the conjugation of dopamine with β-alanine to produce NBAD, a precursor of the yellow pigment. We generated two loss-of-function mutants for Gb'ebony (Gb'ebony cr1 and Gb'ebony cr2 ) using CRISPR/Cas9 genome editing and revealed the role of Gb'ebony in the cuticle pigmentation of crickets. The complete loss-offunction of Gb'ebony in the mutants was verified at the protein level (Fig 4C). Mutations in Gb'ebony resulted in the darkening of the yellow-colored body regions, except for the spotted patterns on the forewing (Fig 5). The darkening of the body color in Gb'ebony may be due to an over-accumulation of dopamine derived from the excess of intracellular dopamine resulting from disrupted NBAD synthesis. Our data clearly demonstrate that the yellow pigment on the cuticle of G. bimaculatus is NBAD yellow sclerotin produced by Gb'ebony, and knockout of Gb'ebony results in the systemic darkening of body color. Similarly, the role of ebony in the cuticle pigmentation of Drosophila results in the systemic darkening of body color, as demonstrated in a genetic ebony knockout strain [14]. In adult Oncopeltus and Periplaneta, depletion of ebony results in the systemic darkening of body color, although in Oncopeltus, the color of the hindwing remains unchanged [17]. Similar body regions are affected by Gb'ebony mutations in Periplaneta and Drosophila. Overall, these results indicate that the systemic function of ebony in cuticle pigmentation is conserved in a broad range of insect species.
In this study, the Gb'ebony mutants displayed a white spotted pattern in the forewing. The white pigment is probably NADA sclerotin. In the absence of Gb'ebony, the NADA and melanin pathways compete because both use dopamine as their substrate. Therefore, there are two possible mechanisms for the formation of a white spotted pattern in the forewing. First, the expression of the melanin synthesis gene yellow is lost in the spots; and second (which we believe is more likely), the expression of the NADA synthesis gene nat is enhanced. The results of recent studies on other insects provide support for the second possibility. For example, aaNAT is highly expressed in the white spots of the hemelytra of Platymeris. RNAi knockdown of this gene results in the blackening of these spots [26]. These results suggest that the expression of the pigment metabolism gene in the forewing spots of Gb'ebony mutants is regulated differently than other body parts, and aaNAT is probably required for the white-spotted patterning in crickets. However, the mechanism behind the spatial expression of pigment metabolism genes remains unknown.

Role of Gb'tan in cuticle pigmentation
Gb'tan encodes NBAD hydroxylase, which catalyzes the degradation of NBAD into dopamine and β-alanine, an activity that is opposite to that of the Gb'ebony gene product. We predicted that Gb'tan knockout would enhance NBAD yellow sclerotin synthesis. We then generated loss-of-function mutants for Gb'tan (Gb'tan cr1 ) to test our prediction. In such mutants, the body color of late-stage nymphs turned a bright yellow color (Fig 6), which is probably due to an over-production of NBAD yellow sclerotin. Our data indicate that Gb'tan is required for proper melanization and that its knockout results in enhanced NBAD yellow sclerotin synthesis. The level of the contribution of the tan gene to cuticle pigmentation in different insect species varies. In Drosophila, the reduction in melanization of tan mutants spans the entire body [18,27]. In contrast, Oncopeltus adults with depleted tan via RNAi display no significant alteration in body color, although the depletion of ebony results in the darkening of body color [17]. Liu et al. (2016) [17] proposed that in hemimetabolous insects, either ebony or tan is required for cuticle pigmentation. Our results mostly agree with this, as the body color of Gb'tan mutants clearly changed only at the seventh nymph stage. However, the reason body color changed during these stages remains unknown. The seventh instar stage shows a relatively low Gb'tan expression (Fig 3B), implying that factors other than Gb'tan expression level may also be involved in the increased appearance of the knockout phenotype.
One possibility is that differences in NBAD levels in vivo at various developmental stages may be involved in the knockout phenotype of Gb'tan, since Gb'Tan uses NBAD as a substrate. At the seventh instar stage, Gb'ebony expression reaches approximately peak levels (Fig 3A), implying that NBAD levels may have peaked as well. This means that the contribution of Gb'tan is particularly high at this stage, and the knockout phenotype has emerged. This hypothesis is also supported by data showing that wild-type males express more Gb'ebony in seventh instar nymphs and adults ( Fig 3A) and that males of Gb'tan mutants at this stage have a more clearly observable phenotype than females (Fig 6). The possibility that ebony function contributes to differences in NBAD levels in vivo during development has been suggested in other insect species; Ze et al. (2022) [16] reported that NBAD may be more abundant in adults of H. vigintioctopunctata.

The mechanism determining body color in Gryllus
In this study, we investigated the function of Gb'ebony and Gb'tan in the metabolism of dopamine and NBAD by generating homozygous knockout mutants through CRISPR/Cas9 genome editing. The body color of Gb'ebony mutants showed a systemic darkening, which was probably the result of dopamine-melanin produced from excess dopamine, indicating that the loss-of-function of Gb'ebony results in enhanced dopamine-melanin synthesis. Meanwhile, the body color of the Gb'tan mutants was yellow, but the function of Gb'tan was not systemic. The yellow pigment is probably NBAD sclerotin, indicating that the loss-of-function of Gb'tan enhances the synthesis of NBAD sclerotin. As shown in Fig 7A, the results of our study indicate that the body color of crickets is generated by three cuticle pigments (dopamine-melanin, NBAD sclerotin, and NADA sclerotin), which are synthesized from dopamine. The dysfunction of one gene in this metabolic pathway caused a body color change due to an over-production of the other pigments.
A summary of the expression profiles of Gb'ebony and Gb'tan from eggs through adults is shown in Fig 7B. In first instar nymphs, Gb'ebony is upregulated after hatching, followed by low expression levels in the second to fourth instar nymphs. Then, the expression levels change periodically in the fifth instar stage through the adults, showing peak levels immediately after molting in each stage. Gb'tan is also expressed according to hatching, and its peak expression levels gradually decrease toward the adult stage. Cricket body colors also change through the stages: in the first instar nymph and the fifth instar to adult, the body color is relatively light, whereas in the second to fourth instar, the body color is relatively dark (Fig 7B). The dark pigmentation is thought to be caused by the accumulation of dopamine-melanin and the depletion of NBDA due to the low and high expression of Gb'ebony and Gb'tan, respectively. However, the light pigmentation probably results from the depletion of dopamine-melanin and the accumulation of NBAD due to high and low expression of Gb'ebony and Gb'tan, respectively. Our results also show that adult male crickets tend to be more yellow than females (Fig 5A). The Gb'ebony expression level was significantly higher in adult males than in females (Fig 3A), suggesting that the yellow color in male crickets is probably NBAD sclerotin that is generated by the high expression of Gb'ebony.
This study provides evidence that the combination of Gb'ebony and Gb'tan expression levels governs the generation of stage-specific body color patterns during postembryonic development. This finding provides new insights into the molecular mechanisms by which insects diversify their body coloration between and within species and adapt to their surroundings.

Animals
Nymphs and adults of the G. bimaculatus white-eyed mutant strain [28] were reared in plastic cases at 30˚C ± 1˚C and 30%-40% relative humidity under a 10 h light and 14 h dark photoperiod. They were fed artificial fish food (Kyorin, Hyogo, Japan).

cDNA cloning
Total RNA was prepared using TRIzol Reagent (Thermo Fisher Scientific, Massachusetts, USA) and treated with ezDNase Enzyme (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA was then reverse transcribed using a SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific) with oligo dT primers. Gb'ebony and Gb'tan cDNA, including the full-length coding sequences, were amplified from cDNA derived from third instar nymphs and adult forewings, respectively, using the primers listed in S1 Table. The PCR products were inserted into the pGEM T-Easy vector (Promega, Wisconsin, USA) and sequenced.

CRISPR/Cas9-mediated genome editing
Preparation of gRNAs and injection solution. The Alt-R CRISPR Cas9 System (IDT, Iowa, USA) was used according to the manufacturer's instructions. CRISPR RNAs (crRNAs), which are specific for target genome sequences, were designed using the online tool CRISPRdirect (https://crispr.dbcls.jp) following the NGG protospacer-adjacent motif (PAM) [29]. The target sequences of the designed crRNAs are listed in S2 Table. The sequence specificity of the crRNAs was checked using the BLASTN program against G. bimaculatus genomic data (Gen-Bank: GCA_017312745.1). The sequences of the designed crRNAs are listed in S2 Table. Each crRNA and trans-activating crRNAs was hybridized in duplex buffer (IDT) at a concentration of 29.7 μM. The 29.7 μM gRNA complex was then mixed with an equal volume of 12.4 μM Cas9 (Alt-R 1 S.p. HiFi Cas9 Nuclease V3; IDT) in the injection solution (1.4 mM NaCl, 0.07 mM Na 2 HPO 4 , 0.03 mM KH 2 PO 4 , and 4 mM KCl) and incubated for 15 min at room temperature. The mixture containing the Cas9-gRNA complex was then diluted 20-fold with injection solution and used for microinjection.
Microinjection into cricket eggs. Cricket eggs were microinjected according to previously reported methods [4,30]. Briefly, the Cas9-gRNA complex was injected into cricket eggs (100-200 eggs) using a microinjector (IM-300 Microinjector; Narishige, Tokyo, Japan) and a compressor (0.2LE-8SBZN, Narishige). Injections were performed within 3 h after starting egg incubation. After the injections, the eggs were soaked in PBS containing penicillin and streptomycin and kept at 30˚C for 2 d. Thereafter, the eggs were transferred on a paper towel that was constantly wetted with water and kept there until they hatched.
Genotyping. Genomic DNA was extracted from eggs or legs using the CicaGeneus Total DNA PrepKit (Kanto-chemical, Tokyo, Japan). Mutations at the gRNA-targeted-regions of Gb'ebony and Gb'tan in the G0 and G1 embryos were detected by amplifying these regions and digesting the PCR products with the Guide-it Mutation Detection Kit (Takara Bio, Japan) based on a previously reported method [13,30]. Briefly, the PCR products were reannealed under the following conditions: 95˚C for 5 min, 95˚C-85˚C at −2˚C/sec, and 85˚C-25˚C at −0.1˚C/sec. Next, 10 μL of the PCR products were mixed with 5 μL of distilled water and 1 μL of Guide-it Resolvase (Takara Bio) in the Guide-it Mutation Detection Kit (Takara Bio). This was incubated at 37˚C for 15 min. Then, mutagenesis in the gRNA-targeted region was detected by electrophoresis using 3% agarose gel. Alternatively, sequence analysis of the gRNA-targeted-regions in the individual G1 and G2 nymphs was performed. The primers used are listed in S1 Table. RT-qPCR All crickets were sampled during the dark period. Unpigmented and pigmented crickets on day 1 of each stage were sampled within 1 h and between 2-15 h, respectively, after hatching and molting. mRNA extraction and reverse transcription were performed from embryos, nymphs, and adult whole bodies according to the method described above (the cDNA cloning section). RT-qPCR was performed in 20 μL reaction mixtures, each containing 10 ng of template cDNA, 0.3 μM of the primers listed in S1 Table, and TB green Premix Ex TaqII (Tli RNaseH Plus; Takara Bio). RT-qPCR was performed using the QuantStudio3 Realtime PCR System (Thermo Fisher Scientific) with Gb'β-actin (Ishimaru et al., 2016) as an internal control. The amounts of Gb'ebony and Gb'tan mRNA were calculated according to the ΔΔCt method.

Image acquisition and intensity measurement
Digital images of nymphs, adults, and isolated adult wings were taken using a DFC7000 T digital camera (Leica, Wetzlar, Germany) connected to an M165 FC stereomicroscope (Leica). Signal intensity, measured as the mean grayscale value on 8-bit images, was determined using the image processing software ImageJ Fiji (https://fiji.sc).

Production of Gb'Ebony and Gb'Tan antibodies
To construct expression vectors of the His-tagged Gb'Ebony and Gb'Tan proteins in Escherichia coli, the coding sequences of Gb'ebony and Gb'tan were amplified using the primers listed in S1 Table and inserted into the Nde I site of the pET41-b vector using In-Fusion Snap Assembly Master Mix (Takara Bio). E. coli strain BL21(DE3) was transformed with the expression vector and was then grown in Luria-Bertani medium containing kanamycin until the optical density at 600 nm reached 0.6. At this point, the expression of the recombinant protein was induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside to the culture and incubating it for 4 h at 37˚C. The cells were collected by centrifugation and resuspended in BugBuster (Merck Millipore, Massachusetts, USA) containing 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF)). The suspension was incubated for 15 min on ice, and the insoluble proteins were collected by centrifugation and dissolved in SDS sample buffer (62.5 mM Tris-HCl (pH6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue). A rabbit and mouse were then immunized with the purified recombinant Gb'Ebony and Gb'Tan protein, respectively.
were performed using the genome of the Gb'tan mutant as a template and the primers listed in S1 Table compared with